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FEZ1) in PERIPHERAL BLOOD REVEALS DIFFERENCES in GENE EXPRESSION in PATIENTS with SCHIZOPHRENIA Vachev TI1,2, Stoyanova VK2,*, Ivanov HY2, Minkov IN1, Popov NT3

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FEZ1) in PERIPHERAL BLOOD REVEALS DIFFERENCES in GENE EXPRESSION in PATIENTS with SCHIZOPHRENIA Vachev TI1,2, Stoyanova VK2,*, Ivanov HY2, Minkov IN1, Popov NT3 18 (1), 2015 l 31-38 DOI: 10.1515/bjmg-2015-0003 ORIGINAL ARTICLE INVESTIGATION OF FASCICULATION AND ELONGATION PROTEIN ζ-1 (FEZ1) IN PERIPHERAL BLOOD REVEALS DIFFERENCES IN GENE EXPRESSION IN PATIENTS WITH SCHIZOPHRENIA Vachev TI1,2, Stoyanova VK2,*, Ivanov HY2, Minkov IN1, Popov NT3 *Corresponding Author: Associate Professor Vili K. Stoyanova, M.D., Ph.D., Department of Pediatrics and Medical Genetics, Medical University ‒ Plovdiv, 15A Vasil Aprilov St., 4000 Plovdiv, Bulgaria. Tel: +359-32- 602-431; Fax: +359-32-602-593. E-mail: [email protected] ABSTRACT peripheral blood of patients with schizophrenia. Our results suggested a possible functional role of FEZ1 Schizophrenia (SZ) is a chronic neuropsychiatric in the pathogenesis of schizophrenia and confirmed disorder characterized by affective, neuromorpho- the utility of peripheral blood samples for molecular logical and cognitive impairment, deteriorated social profiling of psychiatric disorders including schizo- functioning and psychosis with underlying molecular phrenia. The current study describes FEZ1 gene ex- abnormalities, including gene expression changes. pression changes in peripheral blood of patients with Observations have suggested that fasciculation and schizophrenia with significantly down-regulation of elongation protein ζ-1 (FEZ1) may be implicated FEZ1 mRNA. Thus, our results provide support for in the pathogenesis of schizophrenia. Nevertheless, a model of SZ pathogenesis that includes the effects our current knowledge of the expression of FEZ1 in of FEZ1 expression. peripheral blood of schizophrenia patients remains Keywords: Fasciculation and elongation protein unclear. The purpose of this study was to identify ζ-1 (FEZ1); gene expression; quantitative reverse- the characteristic gene expression patterns of FEZ1 transcriptase polymerase chain reaction (qRT-PCR); in peripheral blood samples from schizophrenia pa- schizophrenia (SZ) tients. We performed quantitative reverse-transcrip- tase (qRT-PCR) analysis using peripheral blood from INTRODUCTION drug-free schizophrenia patients (n = 29) and age and gender-matched general population controls (n = 24). Schizophrenia (SZ) is a severe mental disorder For the identification of FEZ1 gene expression pat- affecting approximately 1.0% of the human popula- terns, we applied a comparative threshold cycle (CT) tion worldwide [1]. Twin, family and adoption studies method. A statistically significant difference ofFEZ1 strongly support that genetic factors play an important mRNA level was revealed in schizophrenia subjects role in the etiology of SZ. Currently, numerous genetic compared to healthy controls (p = 0.0034). To the studies, including linkage scans and their meta-analy- best of our knowledge, this study is the first describ- ses, candidate gene association analyses, gene expres- ing a down-regulation of FEZ1 gene expression in sion and genome-wide association studies (GWAS) have identified particular genes and chromosomal loci with the disorder [2-4]. Circulating blood is eas- 1 Department of Plant Physiology and Molecular Biology University ily accessible material and has been suggested as an of Plovdiv “Paisii Hilendarski”, Plovdiv, Bulgaria alternative to tissue samples for molecular profiling of 2 Department of Pediatrics and Medical Genetics, Medical University – Plovdiv, 4000 Plovdiv, Bulgaria human disease and disease risk [3,5]. This is based on 3 State Psychiatry Hospital Pazardzk, Pazardzk, Bulgaria the capacity of peripheral blood to reflect pathological 31 FEZ1 EXPRESSION IN SCHIZOPHRENIA changes in the body. As the brain tissue is not easily International Neuropsychiatric Interviews were done accessible for investigation, blood-based expression by a certified psychiatrist to evaluate the diagnosis profiling is increasingly being undertaken to search of paranoid SZ only, on Diagnostic and Statistical potential biomarkers for SZ [6,7]. Fasciculation and Manual of Mental Disorders (IVth edition) criteria elongation protein ζ-1 (FEZ1) is one of the first identi- and to exclude any mental disorder in the controls. fied binding partners of Disrupted-in Schizophrenia Important inclusion criteria were that the participants 1 (DISC1), a susceptibility gene for major mental had not received any medication (even psychotro- disorders including SZ in yeast two-hybrid screen of pic) 1 month before blood sampling and they had a an adult human brain library [8]. Moreover, proteomic standard breakfast, so they were assessed in a state techniques revealed that FEZ1 protein interacts with of exacerbation. Persons with other chronic medical various intracellular partners, such as motor signal- and current acute somatic/neurologic illness, alcohol ing, and structural proteins, one of which is DISC1 or drug abuse/dependancy were also excluded. The [9]. Currently, the role of FEZ1 in mammalian neuro- sample population included 15 males/14 females di- nal development in vivo is not well understood. The agnosed with SZ and 24 age- and gender-matched FEZ1 null mice exhibit hyperactivity and enhanced general population controls (12 males/12 females) responsiveness to psychostimulants [10], supporting with no evidence for any psychiatric or neurological a po-tential contribution of FEZ1 dysfunction to SZ. disorder in first-grade relatives (Table 1). Recently, additional evidence based on sequencing Table 1. Age descriptive statistics. of DISC1-interacting partner genes including FEZ1 revealed an increased burden of rare missense variants Groups n Mean SD SE Mean in SZ susceptibility in an isolated northern Swedish SZ 29 45.62 12.565 2.333 population [11]. In contrast, the FEZ1 gene shows no Controls 24 46.38 12.673 2.587 association to SZ in Caucasian or African American SD: standard deviation; SE: standard error; SZ: schizophrenia. populations [12]. However, no association was found in either population between specific haplotypes and Blood Collection and RNA Isolation. Blood any of the psychiatric disorders [13]. Interestingly, samples from the patients and control groups were there is a significant reduction of FEZ1 mRNA in collected in PAXgene Blood RNA collection tubes both hippocampus and dorsolateral prefrontal cortex (PreAnalyticX GmbH, Hombrechtikon, Switzerland) of SZ patients and also an association of the DISC1 that contain a reagent that lyses blood cells and im- genotype and FEZ1 mRNA levels [9]. All these find- mediately stabilizes intracellular RNA to preserve the ings raise the possibility that FEZ1 and DISC1 may gene expression profile. To reduce any potential bias assist in regulating both neuronal development and in gene expression due to diurnal variation, blood was risk for SZ. We hypothesized that previously shown drawn in the morning, from all of the subjects. We FEZ1 mRNA levels in prefrontal cortex of SZ patients used the PAXgene Blood miRNA Kit (PreAnalyticX) may be relevant to those in peripheral blood tissue. to extract total RNA from the blood samples [14]. To- The aim of this study was to identify gene expression tal RNA was then quantified by absorbance at A260 patterns of FEZ1 in peripheral blood samples from nm using Epoch Micro-Volume Spectrophotometer SZ patients. System (BioTek, Winooski, VT, USA) and the purity was estimated by the ratio A260/A280 nm. The ab- MATERIALS AND METHODS sorbance ratio of 260 nm and 280 nm (A260/A280) was between 1.93 and 2.1 for all samples included for Ethics Statement. This study and the informed further analysis. The RNA integrity was confirmed consent forms were approved by the Medical Uni- by non denaturing agarose gel electrophoresis, which versity of Plovdiv Ethics Committee. was then stored at ‒80 °C until further analysis. The Participants. Written informed consent was resulting RNA was treated with RNase-free DNase I obtained from 29 patients recruited at the State Psy- (Promega BioSciences, San Luis Obispo, CA, USA) chiatry Hospital Pazardjik, Pazardzk, Pazardzk, Bul- according to the manufacturer’s protocol and checked garia and 24 healthy volunteers. Routine psychiatric for DNA contaminations prior to copy DNA synthesis examination, wide medical hystory and the Mini- step.PAGE 32 BALKAN JOURNAL OF MEDICAL GENETICS Vachev TI, Stoyanova VK, Ivanov HY, Minkov IN, Popov NT Quantitative Reverse-Transcription Poly- the test sample and the calibrator samples was made merase Chain Reaction (qRT-PCR) Analysis of using the equation: FEZ1 mRNA Level. The qRT-PCR analyses were Ct(test) = Ct(target, test)‒Ct(reference, test) performed in at least three physically separate rooms Ct(calibrtator) = Ct(target, calibrator)‒Ct(reference, calibrator) in to order to reduce the chance for contamination. Second, normalization of the Ct of the test sam- Copy DNA for the protein coding gene was synthe- ple to the Ct of the calibrator was made using the sized from total RNA with oligo (dT)18 primer using equation: RevertAid First Strand cDNA Synthesis Kit accord- Ct = Ct(test)‒Ct(calibrator) ing to the assay protocol (Thermo Fisher Scientific, Finally, calculation of the expression ratio with Waltham, MA, USA). Reverse transcription reactions the following equation: contained 1 µg of total RNA samples, 1 µL oligo 2‒Ct = normalized expression ratio. (dT)18 primer and nuclease free water to a final vol- The result obtained is the fold increase (or de- ume of 12 µL, after incubation at 65 °C for 5 min., crease) of the target gene in the test samples relative we added 4 µL 5X RT buffer, 1 µL RiboLock RNase to the calibrator sample. Inhibitor (20 U/µL) (Thermo Fisher
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